In Vitro Reconstitution of the Clostridium botulinum Type D Progenitor Toxin

Kouguchi, Hirokazu; Watanabe, Toshihiro; Sagane, Yoshimasa; Sunagawa, Hiroyuki; Ohyama, Tohru

doi:10.1074/jbc.m106762200

Cited by 54 publications

(42 citation statements)

References 34 publications

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“…Quantitative data concerning NAPs in 12S and 16S progenitor toxins are available for serotype D, strain 4947 (16). For this serotype, four heterodimers of HA-33/HA-17 are present in both 12S and 16S progenitor toxins.…”

Section: Discussionmentioning

confidence: 99%

“…Neurotoxin and NTNH comprise the 12S (M) progenitor toxin (30), the 16S (L) progenitor toxin is composed of neurotoxin, NTNH, and additional NAPs (10,15,19), and the 19S (LL) progenitor toxin is a dimer of the 16S (L) progenitor toxin. Genes encoding progenitor toxins are clustered, have a defined spatial order, and are expressed in a coordinated manner, and progenitor toxin assembly apparently proceeds in a concerted fashion (6,7,14,16,18). Functions ascribed to NAPs range from neurotoxin protection against acidic pH levels and proteases by NTNH to HA-33 assistance in binding to and translocating progenitor toxin(s) through alimentary epithelial barriers (11,12,24).…”

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confidence: 99%

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Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

Hines

Lebeda

Hale

et al. 2005

Appl Environ Microbiol

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Botulinum toxin analysis has renewed importance. This study included the use of nanochromatographynanoelectrospray-mass spectrometry/mass spectrometry to characterize the protein composition of botulinum progenitor toxins and to assign botulinum progenitor toxins to their proper serotype and strain by using currently available sequence information. Clostridium botulinum progenitor toxins from strains Hall, Okra, Stockholm, MDPH, Alaska, and Langeland and 89 representing serotypes A through G, respectively, were reduced, alkylated, digested with trypsin, and identified by matching the processed product ion spectra of the tryptic peptides to proteins in accessible databases. All proteins known to be present in progenitor toxins from each serotype were identified. Additional proteins, including flagellins, ORF-X1, and neurotoxin binding protein, not previously reported to be associated with progenitor toxins, were present also in samples from several serotypes. Protein identification was used to assign toxins to a serotype and strain. Serotype assignments were accurate, and strain assignments were best when either sufficient nucleotide or amino acid sequence data were available. Minor difficulties were encountered using neurotoxin-associated protein identification for assigning serotype and strain. This study found that combined nanoscale chromatographic and mass spectrometric techniques can characterize C. botulinum progenitor toxin protein composition and that serotype/strain assignments based upon these proteins can provide accurate serotype and, in most instances, strain assignments using currently available information. Assignment accuracy will continue to improve as more nucleotide/amino acid sequence information becomes available for different botulinum strains.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

Hines

Lebeda

Hale

et al. 2005

Appl Environ Microbiol

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“…The HA-33⅐HA-17 subcomponent complex, which could not be separated as a pure component during the isolation procedure, was isolated from L-TC using 4 M guanidine as a denaturant, as previously described (17). Ultracentrifugal analysis using sedimentation equilibrium yielded a calculated molecular weight of 77,400 Ϯ 1,700 Da.…”

Section: Resultsmentioning

confidence: 99%

“…Isolation of the HA-33⅐HA-17 complex from 650-kDa L-TCIsolation of the HA-33⅐HA-17 complex from a 250-mg pellet resulting from ammonium sulfate precipitation of the 650-kDa L-TC was performed as described (17).…”

Section: Methodsmentioning

confidence: 99%

A Novel Subunit Structure of Clostridium botulinum Serotype D Toxin Complex with Three Extended Arms

Hasegawa

Watanabe

Suzuki

et al. 2007

Journal of Biological Chemistry

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105

108

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The botulinum neurotoxins (BoNTs) are the most potent toxins known in nature, causing the lethal disease known as botulism in humans and animals. The BoNTs act by inhibiting neurotransmitter release from cholinergic synapses. Clostridium botulinum strains produce large BoNTs toxin complexes, which include auxiliary non-toxic proteins that appear not only to protect BoNTs from the hostile environment of the digestive tract but also to assist BoNT translocation across the intestinal mucosal layer. In this study, we visualize for the first time a series of botulinum serotype D toxin complexes using negative stain transmission electron microscopy (TEM). The complexes consist of the 150-kDa BoNT, 130-kDa nontoxic non-hemagglutinin (NTNHA), and three kinds of hemagglutinin (HA) subcomponents: 70-kDa HA-70, 33-kDa HA-33, and 17-kDa HA-17. These components assemble sequentially to form the complex. A novel TEM image of the mature L-TC revealed an ellipsoidal-shaped structure with "three arms" attached. The "body" section was comprised of a single BoNT, a single NTNHA and three HA-70 molecules. The arm section consisted of a complex of HA-33 and HA-17 molecules. We determined the x-ray crystal structure of the complex formed by two HA-33 plus one HA-17. On the basis of the TEM image and biochemical results, we propose a novel 14-mer subunit model for the botulinum toxin complex. This unique model suggests how non-toxic components make up a "delivery vehicle" for BoNT.Different strains of Clostridium botulinum produce seven distinct serotypes of neurotoxins (BoNTs), 2 classified A through G. BoNT has attracted much interest in recent years due to extensive research on its biochemistry, determination of its crystal structure, and investigations into the pharmacology and applications of BoNTs as therapeutic agents for the treatment of human disease (1-3). After ingestion of BoNT, the BoNT is absorbed from intestinal epithelial cells into the bloodstream, after which it consequently reaches the neuromuscular junctions. BoNT enters nerve cells via receptor-mediated endocytosis, where it cleaves specific sites on target proteins, inhibiting release of neurotransmitters from peripheral cholinergic synapses through its zinc protease activity (4 -6). This process causes muscular paralysis in humans and animals, leading to the disease botulism.Toxins with serotypes A-D and G are encoded by two gene clusters in close proximity to each other; cluster 1 contains the bont and ntnha genes, and cluster 2 contains three genes : ha-70, ha-33, and ha-17 (7, 8). Therefore, botulinum TC consists of five components: BoNT, non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, HA-33, and HA-17). All serotypes of BoNT associate non-covalently with auxiliary non-toxic proteins, thereby forming large toxin complexes (TCs). Serotype A-D strains produce the M-TC (BoNT⅐NTNHA complex) and L-TC (BoNT⅐NTNHA⅐HAs complex) in the culture medium, while serotype E and F strains produce only M-TC. The major biological function of t...

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“…Several different structural models of the fully assembled PTC have been proposed Inoue et al 1996;Kouguchi et al 2002;Lietzow et al 2008;Mutoh et al 2003;Suzuki et al 2005). The discrepancies among the models are largely due to the lack of systematic biochemical and biophysical characterization of HAs in solution.…”

Section: The Structure Of the Ha Proteinsmentioning

confidence: 99%

Assembly and Function of the Botulinum Neurotoxin Progenitor Complex

Gu¹,

Jin²

2012

Current Topics in Microbiology and Immunology

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Botulinum neurotoxins (BoNTs) are among the most poisonous substances known to man, but paradoxically, BoNT-containing medicines and cosmetics have been used with great success in the clinic. Accidental BoNT poisoning mainly occurs through oral ingestion of food contaminated with Clostridium botulinum. BoNTs are naturally produced in the form of progenitor toxin complexes, which are high molecular weight (up to ~900 kDa) multi-protein complexes composed of BoNT and several non-toxic neurotoxin-associated proteins (NAPs). NAPs protect the inherently fragile BoNTs against the hostile environment of the gastrointestinal tract and help BoNTs pass through the intestinal epithelial barrier before they are released into the general circulation. These events are essential for ingested BoNTs to gain access to motoneurons, where they inhibit neurotransmitter release and cause muscle paralysis. In this review, we discuss the structural basis for assembly of NAPs and BoNT into the progenitor toxin complexes that protect BoNT and facilitate its delivery into the bloodstream.

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In Vitro Reconstitution of the Clostridium botulinum Type D Progenitor Toxin

Cited by 54 publications

References 34 publications

Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

A Novel Subunit Structure of Clostridium botulinum Serotype D Toxin Complex with Three Extended Arms

Assembly and Function of the Botulinum Neurotoxin Progenitor Complex

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